Study On The Effects Of Norepinephrin And IL-18 In Acute Stress Aggravating Murine Allergic Contact Dermatitis

Objective To explore the effects of acute restraint stress on murine allergic contact dermatitis.Methods Female BALA/c mice were randomly assigned to three groups:dermatitis group, dermatitis+stress group and challenge only group. On day 0, mice of dermatitis group were shaved on the abdomen. On day 1 and 2 of the experiment,25μl of 0.5%(W/V) DNFB in acetone/olive oil (4:1; AOO) was slowly applied to the abdomen with a micropipette. On day 6,25μl of 0.25% DNFB was pipetted onto the right ear and 25μl of AOO onto the left ear for negative control. All chemicals were applied to the abdomen or the ear at 12 noon. Ear thickness was measured for each ear before hapten application with a micrometer. Measurements were repeated in 24 hours after challenge. Ears from mice were immersion fixed in formaldehyde and embedded in paraffin. Blocks were transversely serially sectioned at 4μm. All sections were stained by standard protocols for haematoxylin and eosin (H&E). Mice of dermatitis+stress group were sensitized on the abdomen with 0.5% DNFB and challenged on the ear with 0.25% DNFB. Two hour restraint was applied prior to challenge on day 6. Mice of challenge only group were challenged on the ear with 0.25% DNFB on day 6 only. Mice of dermatitis group and dermatitis+stress group were evaluated anxiety behavior by elevated plus maze test after challenge.Results Mice of dermatitis+stress group showed more serious anxious behavior than dermatitis group in the elevated plus maze test (P<0.05). The mean ear swelling (%baseline) 24 hours after challenge of ACD mice were significantly increased compared to negative control mice (P<0.01). The mean ear swelling (%baseline) 24 hours after challenge of dermatitis+stress group were significantly increased compared to dermatitis group (P<0.05). Histological analysis of challenged skin showed that skin inflammation in stressed mice was more pronounced and comprised augmented vasodilatation, dermal edema, and cellular infiltration of the dermis. The mean ear swelling (%baseline) of dermatitis+stress group negatively correlated with open arm time (%baseline) (OT%).Conclusions Acute restraint stress increases the levels of anxiety in mice. Acute restraint stress applied before challenge aggravates ACD response in mice. The effect of stress on ACD response is correlated to the anxiety behavior of stressed mice. Objective To explore the effects of acute restraint stress on NF-κB p65 DNA-binding activity and IL-18 mRNA expression in spleen lymphocytes of unsensitized or sensitized mice.Methods Mice were randomly assigned to three groups: unsensitized group, sensitized+stress group and sensitized group. Mice of unsensitized group were stressed only. Mice of sensitized+stress group were sensitized by applying 25μl of 0.5% (W/V) DNFB in acetone/olive oil (4:1; AOO) to the abdomen on day 1 and 2, then they were stressed on day 6 and then challenged by applying 25μl of 0.25% DNFB onto the right ear. Mice of sensitized group were sensitized and challenged only. Mice were sacrificed at 1 minute before,10 minutes,60 minutes or 120 minutes after stress or challenge. Lymphocytes were isolated from spleen. TransAM NF-κB p65 Kit (Activemotif, USA) was used to detect NF-κB p65 activation of nuclear protein of murine spleen lymphocytes. Relative IL-18 mRNA expression in the murine spleen lymphocytes was dectected by semiquantitative RT-PCR analysis.Results NF-κB DNA-binding activity in unsensitized mice increased at 10 min after stress (P<0.01), almost returned to baseline at 60min after stress (P>0.05). Acute restraint stress led to a three-fold increase in IL-18 mRNA expression at 60 min after stress in unsensitized mice (P<0.01), and return to baseline at 120 min after stress. NF-κB DNA-binding activity in stressed mice was significantly higher than that in unstressed mice at both 10min and 60min (stressed vs control at 10 min: P< 0.01; stressed vs control at 60min:P<0.01). IL-18 mRNA expression of stressed mice was significantly higher than that of unstressed mice at 60 and 120 minutes after stress. (stressed vs control at 60min:P< 0.01; stressed vs control at 120min:P< 0.01).Conclusions Acute restraint stress increased NF-κB DNA-binding activity and IL-18 mRNA expression in spleen lymphocytes of both unsensitized and sensitized mice. These changes were temple in unsensitized mice and constant in sensitized mice. NF-κB and IL-18 might be potential mediators in enhancing ACD response by acute stress. Objective To explore the influences of Chemical sympathectomy by 6-OHDA on stress effects.Methods Chemical sympathectomy was performed using the neurotoxin 6 hydroxy-dopamine (6-OHDA). Mice received three alternative-day (days 6,4, and 2 before challenge or stress) i.p. injections (100μl) of 200 mg/kg 6-OHDA (Sigma-Aldrich) in PBS containing 0.01% ascorbate, while control mice received vehicle injections on the same injection schedule. Mice of dermatitis+stress+6-OHDA group were induced ACD and stressed as noted earlier. They received three times injections of 6-OHDA. The control of this group received three times injections of solvent. Ear thickness was measured for each ear before hapten application with a micrometer. Measurements were repeated in the 24 hours after challenge. Mice of stress+6-OHDA group were stressed and received injections of 6-OHDA. The control of this group received three times injections of solvent. Mice were sacrificed at corresponding time point as noted earlier. Mice of sensitized+stress+6-OHDA group were sensitized and stress and challenged. They received three times injections of 6-OHDA. The control of this group received three times injections of solvent. Mice were sacrificed at corresponding time point as noted earlier. TransAM NF-κB p65 Kit (Activemotif, USA) was used to detect NF-κB p65 activation of nuclear protein of murine spleen lymphocytes. Relative IL-18 mRNA expression in the murine spleen lymphocytes was detected by semiquantitative RT-PCR analysis.Results 6-OHDA treatment strongly reduced the stress-induced increase in ear thickness (P<0.01).6-OHDA pretreatment reduced the stress-induced increase in NF-κB DNA-binding activity l0min after stress (P<0.01). The stress-induced increase in IL-18 mRNA 60min after stress was reduced by 6-OHDA pretreatment (P<0.01).6-OHDA decreased the stress-induced NF-κB DNA-binding activity in lymphocytes of sensitized mice at l0min and 60min after stress. (P<0.01). 6-OHDA decreased the stress-induced IL-18 mRNA in lymphocytes of sensitized mice at 60min and 120min after stress. (P<0.01).Conclusions Chemical sympathectomy strongly reduced the stress-induced enhancement of ACD response. And chemical sympathectomy could reduce stress-induced expression of IL-18 and activation of NF-κB in mice spleen lymphocytes. The effects of stress on ACD response may be mediated by the sympathetic nervous system.